The present invention relates to methods of detecting the presence or absence of microorganisms in and of validating the sterility of compositions which contain substantial quantities of lipids.
Process validation refers to the concept of demonstrating the efficacy of a particular process through scientific methods. Injectable compositions which are to be administered to humans must be validated for sterility before the product can be safely released to consumers. This is commonly achieved through the use of a standard battery of microbiological tests which are based on the principle of attempting to isolate viable microorganisms from samples of the pharmaceutical product. Generally, this may involve passing the pharmaceutical product through a filter and isolating any microbes which may be present in the composition on the surface of the filter. The filter is then incubated in a microbiological medium and examined for microbial growth. Alternatively, the sample may be assayed for microbial growth by direct inoculation in a growth medium. By performing such assays with controls, scientists are able to demonstrate when the product of interest is free of contaminating microorganisms and therefore safe for distribution to consumers.
The validation of the sterility of lipid-rich compositions has presented particularly intractable problems. These compositions may include microcrystals, liposomes, microdroplets, and other compositions with a high lipid content. These compositions are generally unfilterable because their heavy lipid matrix quickly blocks the pores of the filter or membrane, thereby stopping the flow of liquid. Even the use of heat, emulsifiers, and pH adjustment often fail to convert these compositions into filterable solutions. While such products can sometimes be validated by direct inoculation, this is not feasible when the pharmaceutical or chemical product is itself bactericidal in nature. These problems are particularly acute, and these methods even more unsuitable, in circumstances where the product contains large quantities of antibiotics. Because of these obstacles, it has been thought that compositions which contain microcrystals., liposomes, and microdroplets are unfilterable. Presently, there are no available methods for filtering these compounds and validating their sterility such that they can be confidently distributed to consumers. This problem is particularly acute when the composition contains a bacteriocidal agent. We have observed the surprising result that these compounds can be brought to a filterable state by applying the chemical principles and schemes described herein.
The present invention provides a convenient, easily performed, and reliable method for determining the presence or absence of microorganisms or for validating the sterility of lipid-containing compositions. The method can be economically performed using commonly available chemicals and laboratory equipment within short timeframes to validate the sterility of products of interest. The methods disclosed herein are also suitable for validating the sterility of chemical and pharmaceutical products which contain large quantities-of antibiotics or other bacteriocidal agents.
The present invention provides methods of determining the presence or absence of microbes in a composition containing a lipid. In preferred embodiments, the lipid may be present in the composition in the form of a phospholipid, for example, as lecithin. In particularly preferred embodiments, the lipid may be present in the composition in the form of liposomes, microcrystals, or microdroplets. The composition may also contain a bacteriocidal agent, such as an antibiotic. In preferred embodiments, the bacteriocidal agent may be oxytetracycline, and the microbes may be a type of bacteria.
The method is conducted generally by dissolving the composition containing a lipid-in a diluent solution to form a diluted sample, passing the diluted sample through a filtration device, incubating the microbes which may be present on the filtration device for a time necessary to observe the growth of microbes which may be present, and examining the assay result to determine the presence or absence of microbes. The dissolution step may be accomplished by providing a diluent solution at a pH where the solubility of the composition containing the lipid, or the solubility of a major component of the composition, is enhanced relative to other pH points. In preferred embodiments, the diluent solution may comprise one or more solubilizers, and may also comprise a salt. The solubilizer may be an emulsifier, or a polysorbate (such as polysorbate 20) or sodium lauryl sulfate and the salt may be any water soluble metal/halogen salt. In particularly preferred embodiments, the salt is sodium chloride or potassium chloride.
The composition containing a lipid may be contacted with the diluent solution, such as by pouring or placing the composition into a bottle containing the diluent solution, or vice versa. The composition may be dissolved in the diluent solution, and then passed through a filtration device. The filtration device may be an ordinary nylon filter with pores of 0.2 xcexcm or small enough to retain at least-a portion of the microbes being sought to be determined. The microbes which may be present on the filter may then be incubated for a period of time necessary to observe their growth. For example, the whole filter may be placed in an appropriate culture medium, or the filtration device filled with medium, or the organisms may be removed from the filter and placed into the medium. After incubation of the microbes, the assay result may then be read to determine if organisms were present in the sample.
The present invention therefore provides methods of validating the sterility of compositions containing lipids.
In preferred embodiments, the present invention may be employed for determining the presence or absence of microorganisms in compositions comprising antibiotics suspended in a phospholipid matrix. In preferred embodiments, the antibiotic may be oxytetracycline, the diluent solution may be a base, and the phospholipids may be present in the form of microcrystals, liposomes, or microdroplets. The diluent solution may also contain a salt and an emulsifier.
Liposomes are multilamaellar vesicles. They generally have a lipid bilayer structure which is also layered within the total vesicle structure. Many layers may exist within the liposome.
Microdroplets comprise liquids encased in a layer of lipids. The lipid structure which surrounds and is part of the microdroplet is generally not multi-layered, but is generally a monolayer structure. Microdroplets typically have a diameter of from about 10 xcexcm to about 40 or 50 xcexcm.
Microcrystals are small crystals which usually have a lipid layer (but not a bilayer). They comprise a solid crystal encased in some type of lipid matrix. Microcrystals generally have a diameter of from about 0.5 xcexcm to about 10 or even 20 xcexcm.
By xe2x80x9cbacteriocidal agentxe2x80x9d is meant any composition which has the effect of inhibiting the growth of or destroying bacteria. Bacteriocidal agents may be antibiotics, detergents, or other chemicals (e.g., sodium azide) which inhibit the growth of or destroy bacteria.
By xe2x80x9csolubilizerxe2x80x9d is meant a compound which increases the solubility of another compound or composition.
The methods of the present invention may be broadly applied to lipid-containing compositions. The methods of the present invention may find particular utility for validating the sterility of compositions which are especially high in lipid content such as compositions containing liposomes, microdroplets, or microcrystals. These methods may find particular suitability in the determination of the presence or absence of microorganisms in or for the sterility validation of lipid-containing compounds which also contain highly insoluble antibiotics, for example, microcrystals which contain oxytetracycline. These compositions are provided as examples and are not intended to be limiting. The person of ordinary skill will realize that these principles are broadly applicable for determining the presence or absence of microorganisms or for validating the sterility of a wide variety of lipid-containing compounds. Compositions containing liposomes, microdroplets, and microcrystals have been very difficult or impossible to filter through a membrane with pores small enough to retain microbes which may be present in filtered solution, due to the high lipid content of these compositions. In particular, microcrystals may contain highly insoluble particulate materials which are coated with lipid. The lipid content of these compositions tends to quickly clog the pores of the membrane and stop the flow of liquid through the membrane. When high concentrations of antibiotics or other bacteriocidal compounds are encapsulated in these compositions, they destroy bacteria which may be xe2x80x9cspikedxe2x80x9d into samples for the purposes-of sterility validation controls, thereby negating the validity of the assay.
The principles of the present invention involve contacting the sample to be tested with a diluent solution which will at least partially solubilize the composition. The diluent solution may contain components-or be designed to have physico-chemical characteristics which are directed towards increasing the solubility of one or more components of the sample to be tested. The diluent solution may thus be an acidic or a basic solution, depending on the solubility or other chemical characteristics of the sample compound of interest at particular pH ranges. In a preferred embodiment, a detergent or emulsifier may be included to facilitate the breaking up and solubilization of the lipid matrix, thereby producing a composition in a sufficiently fluid state that it may be passed through a filter, membrane, or other type of filtration device with pores small enough to retain microbes which may be present in the solution. In a preferred embodiment, a combination of detergents and emulsifiers may be used at a concentration of from 1 to 10% w/v. For example, sodium lauryl sulfate (sodium dodecyl sulfate) or other similar emulsifiers of between 4 and 12 carbons in chain length may produce desirable results. Alternatively, polysorbates of between 20 and 80 carbons in chain length may produce desirable results. Microbes which may be captured on the filtration device may then be incubated with a broad spectrum growth medium which may be capable of supporting the growth of any microbes which may be present. In a preferred embodiment, the medium may be capable of supporting reproductive growth of microbes. In particularly preferred embodiments, the medium may be trypticase soy broth (USP), or fluid thioglycolate medium (USP) or another suitable microbe growth medium.
The methods may also be applied to facilitate the handling of lipid-containing compositions for a variety of other purposes as well. For example, the present invention-may be applied to enhancing analytical assays for which it would be useful to solubilize the lipid content of a sample to be tested. The invention may also be useful in assays where it would be advantageous to increase the reactivity of an analyte within a lipid matrix with a chromophore. The present methods may also be applied for enhanced waste disposal, or enhanced spill clean up in which it may be advantageous to solubilize a lipid matrix prior to disposal. The person of ordinary skill will realize that the present invention is also applicable to other applications where solubilization of a lipid rich material is desirable, and that these applications are meant to be within the scope of the present invention.
The methods of the present invention may be applicable to a wide variety of lipid containing compositions with only minor adjustments to the method.
Generally, the methods of the present invention involve consideration of the solubility profile of the particular composition in question. The pH of the composition should be adjusted with consideration to that profile and the composition adjusted to a pH where its solubility or the solubility of a major component of the composition is enhanced relative to other pH values. Solubilizers, such as detergents, emulsifiers, or other solubilizing agents may then be added in appropriate quantities to solubilize the lipid matrix, the compound of interest, or another major component of the composition. Sodium chloride, potassium chloride, or another water soluble metal/halogen salt may also be added since it has been found to further solubilize lipid matrices, such as lecithin. The surprising result of applying the above concepts may be a diluent solution which is capable of solubilizing the composition of interest, and rendering it filterable. The diluted and solubilized composition may then be filtered and the microbes captured on the filter, membrane, or other filtration device incubated and examined for signs of microbial growth in conventional fashion.